Thioesters represent an important class of molecules that readily react with nucleophiles. Thioesters are particularly useful for conjugation and chemoselective ligation reactions. Chemical ligation involves the chemoselective covalent linkage of a first chemical component to a second chemical component. Unique, mutually-reactive functional groups present on the first and second components can be used to render the ligation reaction chemoselective. For example, thioesters are commonly used to direct the chemoselective chemical ligation of peptides and polypeptides. Several different thioester-mediated chemistries have been utilized for this purpose, such as native chemical ligation (Dawson, et al., Science (1994) 266:776-779; Kent, et al., WO 96/34878; Kent, et al., WO 98/28434).
Unfortunately, conventional conditions under which peptide and other thioesters are prepared (Hojo, et al. Pept. Chem. (1992), Volume Date 1991, 29th pp. 115-20; Canne, et al. Tetrahed. Letters (1995) 36: 1217-20; Hackeng, et al. Proc Natl Acad Sci USA. (1999) 96: 10068-73) and in some instances used, are limited to non-nucleophilic synthetic strategies. For example, a problem faced when attempting to make thioester-activated peptides using Nα-9-fluorenylmethyloxycarbonyl (“Fmoc”)-based peptide synthesis is the unwanted destruction of the thioester moiety by strong nucleophiles such as piperidine or piperidine-generated hydroxide ions during synthesis of the peptide. This is a significant problem since the preferred reagent employed to remove Nα-Fmoc groups in each cycle of Fmoc-based organic synthesis contains piperidine. Piperidine like other strongly basic or nucleophilic compounds (hereinafter “nucleophiles”) destroys the thioester component of the peptide, rendering it useless for subsequent thioester-mediated reactions.
Several attempts have been made to address this problem. Clippingdale et al. (J. Peptide Sci. (2000) 6: 225-234) have used a non-nucleophilic base to remove Nα-Fmoc groups of peptides made using Fmoc-based Solid Phase Peptide Synthesis (“SPPS”). This approach has several problems, including generation of unwanted deletions, side-products, and requirement for backbone protection strategies. Other groups, including, Bertozzi et al. (J. Amer. Chem. Soc. (1999) 121:11684-11689) and Pessi et al. (Journal of the American Chemical Society; 1999; 121: 11369-11374.), have reported adapting Fmoc SPPS in combination with a ‘Kenner’ safety-catch linker, which is stable to nucleophiles until the linker has been alkylated, to produce a fully protected peptide-thioester in solution. A drawback of this approach is the poor solubility properties of protected peptides in solution. Other drawbacks of this approach include side reactions inherent to the method, such as the formation of unwanted alkylated byproducts when the linker is alkylated to render it labile, and thus it is impractical for many applications. Similar frustration has been experienced in nucleophilic-based synthesis schemes for molecules other than peptides, such as small organic molecules.
Accordingly, what is needed is a universal and robust system for generating nucleophile-stable thioester-generating compounds and compositions compatible with organic or aqueous reaction conditions for use in various organic synthesis strategies, and conjugation and chemoselective ligation reactions that employ thioester-mediated reactions. The present invention addresses this and other needs.